Mechanistic analysis of the Saccharomyces cerevisiae kinesin Kar3.

The Journal of Biological Chemistry
A T MackeyS P Gilbert

Abstract

Kar3 is a minus-end-directed microtubule motor that is implicated in meiotic and mitotic spindle function in Saccharomyces cerevisiae. To date, the only truncated protein of Kar3 that has been reported to promote unidirectional movement in vitro is GSTKar3. This motor contains an NH2-terminal glutathione S-transferase (GST) tag followed by the Kar3 sequence that is predicted to form an extended alpha-helical coiled-coil. The alpha-helical domain leads into the neck linker and COOH-terminal motor domain. Kar3 does not homodimerize with itself but forms a heterodimer with either Cik1 or Vik1, both of which are non-motor polypeptides. We evaluated the microtubule-GSTKar3 complex in comparison to the microtubule-Kar3 motor domain complex to determine the distinctive mechanistic features required for GSTKar3 motility. Our results indicate that ATP binding was significantly faster for GSTKar3 than that observed previously for the Kar3 motor domain. In addition, microtubule-activated ADP release resulted in an intermediate that bound ADP weakly in contrast to the Kar3 motor domain, suggesting that after ADP release, the microtubule-GSTKar3 motor binds ATP in preference to ADP. The kinetics also showed that GST-Kar3 readily detached fr...Continue Reading

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Citations

Oct 4, 2006·Biochemistry·Jared C CochranSusan P Gilbert
Sep 15, 2012·The Journal of Biological Chemistry·Chun Ju ChenSusan P Gilbert
Aug 19, 2005·The EMBO Journal·Hsiao Mei Annie ChuSharyn A Endow
Jan 25, 2007·Journal of Biochemistry and Molecular Biology·Shiling JiangGuoqin Liu
Nov 10, 2012·Biochemical and Biophysical Research Communications·Caroline DelormeJohn S Allingham
Jul 12, 2005·Yeast
Aug 20, 2010·Physical Biology·Ruizheng Hou, Zhisong Wang

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